Structures and functions of metalloflavoenzymes which produce free radicals

黄嘌呤脱氢酶/氧化酶和硝酸合成酶是产生自由基的复合黄蛋白。在这项研究中，我们展示了二聚体（Mr 290,000）牛奶XDH在2.1 Å分辨率和XO在2.5 Å分辨率下的晶体结构，并描述了XDH在蛋白水解转化为XO形式时发生的主要变化。每个分子由一个含有两个铁硫中心的n端20 kDa结构域、一个中心40 kDa的FAD结构域和一个c端85 kDa的钼铜素结合结构域组成，四个氧化还原中心几乎以线性方式排列。XDH表面暴露环的切割导致靠近黄素环的另一个环的结构重排（jin -423- lys -433）。这种运动部分阻断了NAD底物对FAD辅因子的通路，并改变了活性位点的静电环境，反映了该酶两种形式的底物特异性的切换。我们还构建了几种大鼠肝脏XDH突变酶，并对其性状进行了测定，确定了转化的相关残基。我们用多种物理化学方法表达了小鼠神经元硝酸合成酶（NOS）及其自然突变体的同工型，并测定了酶的特性。我们还研究了其他一些相关的酶，如含血红素的黄素蛋白、纤维二糖脱氢酶和血红素结合蛋白，HBP23具有过氧化物酶活性。

Xanthine dehydrogenase/oxidase and nitric acid synthetase are complex flavoprotein that produce free radicals. In this study, we presented the crystal structure of the dimeric (Mr 290,000) bovine milk XDH at 2.1 Å resolution and XO at 2.5 Å resolution and describe the major changes that occur upon the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20 kDa domain containing two iron sulfur centers, a central 40 kDa FAD domain, and a C-terminal 85 kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gin-423-Lys-433). This movement partially blocks access of the NAD substrate to the FAD cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme. We have also constructed several mutant enzyme of rat liver XDH and determined the character to confirm the responsible residues for conversion.We have expressed mouse neuronal nitic acid synthase (NOS) as well as the iso-form of natural mutant and determined characters of the enzymes by several physicochemical methods. We have also studied some other related enzymes such as heme containing flavo-protein, cellobiose dehydrogenase, and heme binding protein, HBP23 has peroxidase activity.